Computational protocol: Description and DNA barcoding of three new species of Leohumicola from South Africa and the United States

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Protocol publication

[…] Internal Transcribed Spacer (ITS) and Cox1 sequences were aligned using MAFFT v6 (). A few minor adjustments were made to the ITS alignment using Se-Al v. 1.0 (). No manual adjustments were required for the Cox1 alignment, which had no indels. Alignments are deposited in TreeBASE (, study accession no. S2134. To test whether the ITS and Cox1 data sets contained congruent phylogenetic signals and could be combined for analysis, a partition homogeneity test () was performed using PAUP 4.0 () using a heuristic search with 1 000 replicates, TBR branch swapping, unordered and unweighted characters and gaps treated as missing.Parsimony analyses of ITS alone (), Cox1 alone (), and for both genes combined (), were performed using heuristic searches in PAUP 4.0 (), with uninformative characters excluded. Bootstrap analyses (1 000 replicates) were undertaken using full heuristic searches for the two single-gene parsimony analyses, and using fast-stepwise addition for the dual-gene analysis. For all parsimony analyses, parsimony tree scores were calculated and the 70 % consensus tree was computed.For Bayesian analysis, MrModeltest v. 2.2.6 () was used to select the most appropriate models of sequence evolution for data sets that contained Leohumicola species only, according to the Akaike information criterion (AIC) (). The HKY+I model () and the K80+G () model were selected for Cox1 and ITS, respectively. Bayesian inference was performed with MrBayes v. 3.1.2 () using the dual-gene data set with two designated partitions (ITS and Cox1), the appropriate model of sequence of evolution applied to each partition, and L. lenta DAOM 231149 set as outgroup (see below). Two independent MCMC runs were performed simultaneously. Each MCMC ran for 2.0 × 106 generations, sampling every 100 generations, for a total of 20 001 trees. Acceptable convergence was attained after 1.0 × 106 generations, and the first 10 000 trees were discarded as burn-in. The 10 001 trees from each independent MCMC (total 20 002 trees) were combined into one consensus tree with 50 % majority rule consensus (). Myxotrichum deflexum was initially chosen as an outgroup to root all analyses, based on its position as near neighbour to the Leohumicola clade in the 18S analyses by . We could not obtain satisfactory sequences for the Cox1 of two other potential outgroups, Myxotrichum arcticum UAMH 9243 and Scytalidium lignicola DAOM 231160. Therefore, M. deflexum was used as the outgroup for the single-gene parsimony analyses. In the Bayesian analysis, the branch connecting M. deflexum to the ingroup was too long, obscuring the phylogenetic structure of the ingroup. Therefore, L. lenta was used to root the tree for the Bayesian and dual-gene parsimony analyses because of its basal position in the single-gene parsimony analyses.Two Leohumicola isolates (DAOM 239499, 239516) had an intron in the Cox1 region. Internal sequencing primers LHM1R (5′-GGCGTTCTTAGTTCTCCATTTAGT-3′), LHM5F (5′-TTAAGTGGGGTACAAAGTCA-3′) and LHM4F (5′-GGTATAGAAAATGGAGCAGGTA-3′) were designed and used to sequence the poorly resolved region at the ends of the exonic region for Leohumicola sp. DAOM 239499. Similarly internal sequencing primers LHM2R (5′-GGCGTTCTTAGTTTTCCATT-3′), LHM3F (5′-CCGCCTAGTTTATTATTATTTTTA-3′) and LHM4F were used for the intron of Leohumicola sp. DAOM 239516. […]

Pipeline specifications

Software tools MAFFT, Se-Al, PAUP*, MrModelTest, MrBayes
Databases TreeBASE
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Hemisus marmoratus